Previous experience with High Intensity Focused Ultrasound (HIFU) has demonstrated that HIFU induces coagulative necrosis in tumor tissues and controls tumor growth of localized prostate, renal cell and liver cancers. The goal of this proposal is to build on the HIFU treatment of local tumors and combine HIFU with tumor immunotherapy to address metastatic cancer. Immunotherapy with tumor vaccines has the potential to induce a tumor-specific immune response that enables the body's own immune system to target metastatic tumors that are distant from the primary tumor. However, clinical experience with tumor vaccines is less than satisfactory because it is difficult to induce protective immunity against tumor antigens due to immune tolerance to "self" antigens and an immunosuppressive environment induced by the tumor. To investigate the complimentary nature of HIFU with immunotherapy, in collaboration with Naren Sanghvi of Focus Surgery, we constructed a HIFU system that is capable of providing the user with control over the acoustic properties of the probe, such as, the power, frequency, pulse length, and pulse repetition frequency and can be used for animal studies. With this HIFU delivery system, we were able to treat tumors with "high" (5 W Total Acoustic Power and operating frequency of 4Mhz) and "low" (40 W Total Acoustic Power and operating frequency of 1 Mhz) energy HIFU that induces instantaneous thermal (80-95oC) tissue coagulative necrosis or nonlethal, mild hyperthermia, respectively. The overall goal is to induce a non-lethal temperature elevation in tumor cells with "low" energy HIFU to induce HSPs and stimulate the immune response, while "high" energy HIFU would be used to ablate the local tumor and release tumor-derived HSPs and peptides for an in situ auto-vaccination. In a recently completed phase I SBIR proposal (PI, Naren Sanghvi, Focus Surgery), we demonstrated that cycles of Low-HIFU, followed by High-HIFU one day later, repeated at one week intervals for a total treatment of 3 cycles, induced a strong tumor-specific cell-mediated immunity (TH1 and CTLs) against murine prostate cancer cells, resulting in better tumor control. Based upon these results, we hypothesized that exposure of solid tumors to "low" energy HIFU would induce protein denaturation, followed by proteosomal degradation of denatured proteins and an eventual increase in the amount of proteosome-processed, antigenic peptides that are bound to HSPs. Subsequent exposure of the PC tumors to "high" energy HIFU, 1-2 days later, would induce cell death and the release of HSP-peptide complex into the extracellular compartment and into the blood stream, resulting in an autologous in-situ tumor vaccination. Thus, a sequential administration of "low" and "high" energy HIFU would provide a source of tumor cell-derived HSP-peptide complex as tumor antigens for professional antigen presenting cells, such as, dendritic cells, and thereby induce a strong tumor-specific systemic immune response that would augment the efficacy of HIFU to control both local and systemic disease.To examine our hypothesis experiments will be performed with the following specific aims: Specific Aim 1: HIFU-enhanced tumor antigen presentation. To optimize the treatment parameters and determine the HIFU treatment sequence ("Low" Energy HIFU + "High" Energy HIFU) that generates the highest tumor- Specific immune response in murine models of solid tumors (lung, liver and prostate cancer). Specific Aim 2: HIFU-enhanced tumor vaccination. To determine whether HIFU can augment the immune response to tumor vaccines. Tumor cells expressing model tumor antigens, such as, ovalbumin (OVA) and carcinoembryonic antigen (CEA) will be used in these studies. Animals will receive CEA and OVA tumor vaccines, followed by cycles of Low-High HIFU of a flank tumor. Animals will be observed for development of spontaneous metastases and survival following a challenge of intravenous tumor cells. Specific Aim 3: HIFU-chemotherapy-enhanced tumor vaccination. To determine whether a combination of HIFU and systemic chemotherapy can induce tumor antigen release and induce stronger systemic immunity. Furthermore, novel chemotherapeutic agents that suppress STAT3-mediated immunosuppression will be combined to examine whether inhibiting immunosuppressive pathways in tumor can augment the HIFU-induced tumor specific protective immunity. Public Health Relevance Statement (provided by applicant): High intensity focused ultrasound, also known as HIFU, is a noninvasive treatment where cancer cells can be killed with the use of ultrasound. In Europe, Canada and Japan, HIFU is used to treat prostate cancer that has not spread outside the prostate. In this proposal, we plan to use HIFU to treat cancer that has spread out of the primary region. Our goal is to use the heating properties of HIFU with tumor vaccines to stimulate the body's own immune system so that it can fight to eradicate cancer cells throughout the body. Successful completion of these studies will find a way to combine HIFU and cancer vaccines for the fight against cancer.